On-slide detection of enzymatic activities in selected single cells

Publikation: Forskning - peer reviewTidsskriftartikel

DOI

  • Josephine Geertsen Keller
    Josephine Geertsen KellerDepartment of Molecular Biology and Genetics, C. F. Møllers Allé 3, Bldg. 1131, Aarhus University, 8000 Aarhus C, Denmark. brk@mbg.au.dk.
  • Cinzia Tesauro
    Cinzia TesauroDepartment of Molecular Biology and Genetics, C. F. Møllers Allé 3, Bldg. 1131, Aarhus University, 8000 Aarhus C, Denmark. brk@mbg.au.dk.
  • Andrea Coletta
  • Astrid Damgaard Graversen
    Astrid Damgaard GraversenDepartment of Molecular Biology and Genetics, C. F. Møllers Allé 3, Bldg. 1131, Aarhus University, 8000 Aarhus C, Denmark. brk@mbg.au.dk.
  • Yi-Ping Ho
  • Peter Kristensen
  • Magnus Stougaard
  • Birgitta Ruth Knudsen

With increasing recognition of the importance in addressing cell-to-cell heterogeneity for the understanding of complex biological systems, there is a growing need for assays capable of single cell analyses. In the current study, we describe the measurement of human topoisomerase I activity in single CD44 positive Caco2 cells specifically captured from a mixed population on glass slides, which were dual functionalized with anti-CD44-antibodies and specific DNA primers. On-slide lysis of captured CD44 positive cells, resulted in the release of human topoisomerase I, allowing the enzyme to circularize a specific linear DNA substrate added to the slides. The generated circles hybridized to the anchored DNA primers and acted as templates for a solid support rolling circle amplification reaction leading to the generation of long tandem repeat products that were detected at the single molecule level in a fluorescent microscope upon hybridization of fluorescent labelled probes. The on-slide detection system was demonstrated to be directly quantitative and specific towards CD44 positive cells. Moreover, it allowed reproducible detection of human topoisomerase I activity in single cells.

OriginalsprogEngelsk
TidsskriftNanoscale
ISSN2040-3364
DOI
StatusE-pub ahead of print - 5 sep. 2017
Eksternt udgivetJa

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